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An efficient molecular design breeding strategy for grape coloring trait based on MYB haplotypes Le Guan, Songtao Jiu, Haifeng Jia and Jinggui Fang * College of Horticulture, Nanjing Agricultural University, Nanjing, 210095, P.R. China Email:

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XII International Conference on Grapevine Breeding and Genetics 15-20, July 2018. Bordeaux, France

An efficient molecular design breeding strategy for grape coloring trait based on MYB haplotypes

Le Guan, Songtao Jiu, Haifeng Jia and Jinggui Fang*

College of Horticulture, Nanjing Agricultural University, Nanjing, 210095, P.R. China Email: fanggg@njau.edu.cn

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Grapevine is one of the most important fruit trees in China

Grapevine cultivation in China

 Wide distribution  High yields  High economic benefits  Mainly consumed as fresh fruits

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Anthocyanin composition is responsible for the color diversity of grape berries.

Anthocyanin composition and color diversity

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The MYB haplotype is the genetic determinant of grape color

(Kobayashi et al. 2004, Walker et al. 2006, Azuma et al. 2009, 2011)

Chr2 MybA2 MybA1 Color locus Alleles VvmybA1a VvmybA1b VvmybA1c VvmybA1SUB VvmybA1BEN VlmybA1-3* Alleles VvmybA2w VvmybA2r VlmybA1-2* VlmybA2*

Gret1: grapevine retrotransposon1 LTR: long terminal repeat TS: target site for the Gret1 insertion

MybA1 and MybA2, inherited together, can be regarded as the color locus.

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Haplotype composition at the color locus is a major genetic determinant of skin color

(Azuma et al. 2017)

A model of the evolutionary differentiation of MYB haplotypes at the color locus in Vitis species

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Can we achieve early prediction of color diversification according to the MYB haplotype composition? Assisting in breeding of high quality cultivars with favorable coloration

Our goals and questions raised

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Coloration classification of the 213 investigated grape varieties

Numbers of grape varieties

The 213 grape varieties were classified into 7 categories according to the depth of the coloration.

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Identification of MYB haplotype composition of the 213 grape varieties by PCR and sequencing

VvMybA1a VvMybA1BEN 1559 bp 2105 bp VvMybA1b VvMybA1c VvMybA1SUB VlMybA1-3 1675 bp 846 bp 1035 bp + sequencing 999 bp + sequencing

DNA quality control by the amplification of actin

MybA1 allele identification

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VlMybA2 161 bp VlMybA1-2 251 bp

Identification of MYB haplotype composition of the 213 grape varieties by PCR and sequencing

1446 bp + sequencing 1444 bp + sequencing VvMybA2r VvMybA2w

DNA quality control by the amplification of actin

MybA2 allele identification

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MYB haplotype composition of the investigated grape varieties

MYB haplotype composition of 211 grape varieties can be identified by PCR and sequencing except ‘Yuanruihei’ (V. vinifera) and ‘Olarra Queen’ (V. labrusca).

V. vinifera
V. vinifera x V. labrusca

A/E1/E2 A/E1 A/C-Rs/E1 A/C-Rs/E2 A/C-Rs A/C-N A/B A/E2 A/F C-N C-N/E2 C-Rs C-Rs/E1/E2 F G/C-N G/F

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MYB haplotype composition of ‘Yuanruihei’ and ‘Olarra Queen’

MYB haplotype composition of ‘Yuanruihei’ and ‘Olarra Queen’ could be  either A/C-N or G/C-Rs  identified by self-crossing

‘phased phase’ HapA HapC-N ‘repulsive phase’ HapC-Rs HapG

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Code VvMybA1a VvMybA1b VvMybA1c VvMybA1SUB VvMybA1BEN VvMybA2r/w Haplotype composition

1 + 2w A 2 + 2w A 3 + 2w A 4 + 2w A 5 + 2w A 6 + 2w A 7 + 2w A 8 + 2w A 9 + 2w A 10 + + 2w/2r AC-N 11 + + 2w/2r AC-N 12 + + 2w/2r AC-N 13 + + 2w/2r AC-N 14 + + 2w/2r AC-N 15 + + 2w/2r AC-N 16 + + 2w/2r AC-N 17 + + 2w/2r AC-N 18 + + 2w/2r AC-N 19 + + 2w/2r AC-N 20 + + 2w/2r AC-N 21 + + 2w/2r AC-N 22 + + 2w/2r AC-N 23 + + 2w/2r AC-N 24 + + 2w/2r AC-N 25 + + 2w/2r AC-N 26 + 2r C-N 27 + 2r C-N 28 + 2r C-N 29 + 2r C-N 30 + 2r C-N 31 + 2r C-N 32 + 2r C-N 33 + 2r C-N

Haplotype composition of ‘Yuanruihei’ was A/C-N.

Identification of MYB haplotype composition of ‘Yuanruihei’

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Code VvMybA1a VvMybA1b VvMybA1c VvMybA1SUB VvMybA1BEN VvMybA2r/w Haplotype composition

1 + 2w A 2 + 2w A 3 + 2w A 4 + 2w A 5 + 2w A 6 + 2w A 7 + + 2w/2r AC-N 8 + + 2w/2r AC-N 9 + + 2w/2r AC-N 10 + + 2w/2r AC-N 11 + + 2w/2r AC-N 12 + + 2w/2r AC-N 13 + + 2w/2r AC-N 14 + + 2w/2r AC-N 15 + + 2w/2r AC-N 16 + + 2w/2r AC-N 17 + + 2w/2r AC-N 18 + + 2w/2r AC-N 19 + 2r C-N 20 + 2r C-N 21 + 2r C-N 22 + 2r C-N 23 + 2r C-N 24 + 2r C-N 25 + 2r C-N

Identification of MYB haplotype composition of ‘Olarra Queen’

Haplotype composition of ‘Yuanruihei’ was A/C-N.

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Is coloration and haplotype composition closely correlated?

 A total of 8 haplotypes and 19 haplotype compositions were identified;  HapC-N and HapE2 showed stronger effect than HapC-Rs, HapB and HapE1;  And thus VlmybA2 might have stronger effect than Vlmyb1-2 regarding coloration;  The more functional alleles, the darker the skin tended to be.

V. vinifera
V. vinifera x V. labrusca

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Can we achieve early color prediction during cross breeding?

b) The berry color observation and quantification a) Haplotype identification and berry coloration

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‘Muscat Hamberg’ ♀ AC-Rs ‘Crimson seedless’♂ AC-Rs × A/A A/C-Rs C-Rs/C-Rs F1 : : 1 2 1 Expected 18 32 19 Observed a

aChi square value=0.391,

P value=0.82 Berry color

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a) Haplotype identification and berry coloration

Can we achieve early color prediction during cross breeding?

‘Cuibao seedless’♀ A/A ‘Qiuhongbao’ ♂ A/C-Rs × A/A A/C-Rs F1 : 1 1 Expected 25 38 Observed a

aChi square value=2.29,

P value=0.13 Berry color

b) The berry color observation and quantification

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Summary

213 grape varieties Color index MYB haplotype composition Verification Early prediction of color diversification can be achieved by the identification of the MYB haplotype composition  The berry coloration matched with the haplotype composition in 99.1% of the investigated varieties;  AC-N and AE2 tended to show deeper colors;  The more functional alleles it contained, the darker the skin color tended to be ‘Muscat Hamberg’ × ‘Crimson seedless’ Cuibao seedless’ × ‘Qiuhongbao’ Closely correlated Confirmed An efficient molecular design breeding strategy for grape coloring trait based on MYB haplotypes

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Potential strategy for the way forward

Multiple target breeding traits Selection of multiple candidate parents with favorable target traits from the germplasm MYB haplotype database cross-check Preference parents with MYB haplotype generating target color trait More hybrids with desirable target traits including berry color x

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Thank you for your attention!

Acknowledgements

Zhengzhou Fruit Research Institute, CAAS National Natural Science Foundation of China Fundings Ministry of Technology

and Science, P.R. China